Physiological and bioinformatic studies on polarity development in Ceratopteris richardii spores.

摘要

Spores of the fern Ceratopteris richardii exhibit gravity directed polarity development. Previous research has indicated that a polar, bottom-to-top, calcium flux is present during the period of axis alignment and polarity fixation, and that this calcium current reorients within five to ten minutes of reorientation of the spore. We measured the ability of the current to reorient as quickly as 42 seconds after reorientation and found that the bottom-to-top calcium flux had already reoriented within that timeframe. This timeframe places restrictions on the molecular mechanisms of current reorientation, favoring a localized activation model, in which the current components are uniformly distributed in or near the plasma membrane of the spore, and are locally activated in response to the gravity signal. We also examined gene expression during polarity development by sequencing randomly selected clones from a spore cDNA library as expressed sequence tags (ESTs). Analysis of over 5,000 ESTs yielded 3,930 tentative unique genes (TUGS) expressed during spore germination. The identified TUGs represent approximately 25% of the expected total TUG population at this timepoint, indicating that EST sampling is far from complete. Analysis of the EST library also indicated expression of a number of dessication and dormancy related genes specifically expressed in Arabidopsis seeds. Additionally, the EST library was used to identify and clone three calcium signaling related genes, verifying the library as a useful approach to identify genes expressed during spore polarity development in Ceratopteris. Finally, a direct method for induction of RNA Interference (RNAi), the simple addition of dsRNA to the germination medium, was tested. This method has previously proven useful in other fern spore systems, but failed to induce gene silencing in Ceratopteris. Initial and apparently successful results using the approach were subsequently shown to be an artifact of the carryover of exogenous dsRNA into the RNA isolated for reverse transcription PCR assessment of gene expression. These studies have added to our resources for Ceratopteris research and have advanced our understanding of a single celled system in which the alignment of its polar axis is governed by a pervasive external signal, gravity.